Method for evaluating microsatellite instability in a tumor sample

ABSTRACT

A detection method for high throughput screening for tumor microsatellite instability. The method employs a panel of microsatellite loci and it is based on a fluorescent multiplex PCR system. The method provides a fast, sensitive, and cost-effective high throughput screening method of MSI detection. The method allows many samples to be processed in one day on a single polyacrylamide gel, and it utilizes much less nucleic acid sample than conventional methods.

FIELD OF THE INVENTION

[0001] The invention relates to the evaluation of microsatelliteinstability in a tumor sample by detecting microsatellite loci in thesample.

BACKGROUND OF THE INVENTION

[0002] Microsatellite instability (MSI) is defined as the occurrence ofnovel alleles in tumor DNA with a frequency of at least 40% amongmicrosatellite loci examined. Defects in the mismatch repair systemcauses MSI which plays an important role in the development of tumors.MSI was first reported in colorectal tumors (Peinado, M. A. et al, 1992Proc. Natl. Acad, Sci. USA 89: 10065-69; Ionov, Y. Nature (London)363:558-61; Thibodeau, S N. Et al Science 260, 1993 260: 816-819), andlater in several other tumor types (Risinger, J I Cancer Res,1993,53:5100-03; Han, H J et al Cancer Res 1993 Cancer Res 1993,53:5087-89; Peltomaki, P, 1993, 53:5853-55; Gonzalez-Zulueta, M et alCancer Res 1993, 5620-23; Merlo, A et al Cancer Res 1994, 54:2098-2101).MSI in inherited nonpolyposis colorectal carcinoma in patients areapparently due to inherited and somatic mutations in mismatch repairgenes (Leach F et al, 1993, 75: 1215-1225; Fishel R et al, 1993, Cell75: 1027-38; Papadopoulos, N, et al 1994, Science 263: 1625-29, 1994;Bronner, C. E. et al, 1994, Nature (London) 368:258-61 (1994).

[0003] Detection of tumors with MSI has important prognostic andtreatment implications for patients. For example, microsatellite markershave been used for colon cancer detection (Cawkwell et al, 1994, Br. J.Cancer 70:813-18). PCR is used for identifying both the appearance ofnew polymorphisms and the loss of heterozygosity in cancer detection(Mao, L. et al Proc. Natl. Acad. Sci. 1994, 91: 9871-75; Mao, L et al,1996 Science, 271:659-62; Radford, et al Cancer Res., 1995, 55:3399-05).However, PCR has limitations in that each PCR reaction is runindividually and separated on a sequencing gel.

[0004] There is a need for large-scale multiplex methods for detectinglarge numbers of microsatellite loci for practical identification ofindividuals for genetic cancer diagnosis and prognosis.

SUMMARY OF THE INVENTION

[0005] The present inventors have developed a MSI detection method forhigh throughput screening for tumor microsatellite instability. Themethod employs a panel of microsatellite loci and it is based on afluorescent multiplex PCR system and automated fragment analysis. Themethod provides a fast, sensitive, and cost-effective high throughputscreening method of MSI detection. The protocols described herein aresimple enough to be performed in a routine clinical laboratory. Themethod also allows many samples to be processed in one day on a singlepolyacrylamide gel, and it utilizes much less nucleic acid sample (about25ng) than conventional methods.

[0006] Broadly stated the present invention relates to a method forevaluating microsatellite instability in a tumor sample by detectingmicrosatellite loci in the sample comprising:

[0007] (a) amplifying in the sample at least two selected microsatelliteloci associated with cancer to provide labeled amplified products oramplicons that are complementary to microsatellite loci sequences in thetumor sample;

[0008] (b) detecting the labeled amplified products or amplicons anddistinguishing the amplified products to indicate the presence of one ormore of the microsatellite loci in the sample; and

[0009] (c) repeating steps (a) and (b) for at least two differentselected microsatellite loci.

[0010] In an embodiment of the invention the microsatellite loci areamplified using a multiplex polymerase chain reaction.

[0011] In an embodiment, a method is provided for evaluatingmicrosatellite instability in a tumor sample by detecting microsatelliteloci in the sample comprising:

[0012] (a) forming a polymerase chain reaction mixture comprising thetumor sample, a polymerase, and primer sets for at least two selectedmicrosatellite loci associated with cancer, each primer setcharacterized by (a) a forward primer containing a sequencecomplimentary to a 5′ upstream primer-specific portion of a selectedmicrosatellite loci; and (b) a reverse primer complementary to a 3′downstream primer-specific portion of the same microsatelite loci,wherein one of the primers has a detectable reporter label;

[0013] (b) subjecting the polymerase chain reaction mixture topolymerase chain reaction cycles to form amplified productscomplementary to microsatellite loci in the tumor sample;

[0014] (c) detecting the reporter labels and distinguishing theamplified products to indicate the presence of one or more of themicrosatellite loci in the sample; and

[0015] (d) repeating steps (a) to (c) with primer sets for at least twodifferent selected microsatellite loci.

[0016] The invention also contemplates kits comprising compositionsselected from the group consisting of primers and ancillary reagentsused in an amplification reaction (preferably PCR) in a method forevaluating microsatellite instability in a tumor sample.

[0017] The methods ofthe invention may be used to determine a genomicinstability index. The index may be calculated as follows:

[0018] (# alterations in the banding pattern from the amplified tumorcell DNA/total number of bands in the pattern from the amplified normalcell DNA)×100

[0019] The methods of the present invention may be used to detectcancer, particularly cancers involving defects in mismatch repair.Various aspects of the invention may be used to identify defects inmismatch repair of genes in the following human cancers: leukemia,colorectal cancer, breast cancer, lung cancer, prostate cancer, braintumors, central nervous system tumors, bladder tumors, melanomas, livercancer, osteosarcoma and other bone cancers, testicular and ovariancarcinomas, head and neck tumors and cervical cancer.

[0020] The methods of the present invention have particular applicationin the diagnosis and monitoring of colorectal cancer. MSI is observed inapproximately 15-25% of sporadic colorectal cancers and more than 85% ofcolorectal cancers arising in patients with hereditary non-polyposiscolorectal cancer syndrome.

[0021] Therefore, the present invention provides a method for diagnosingcolorectal cancer or hereditary non-polyposis colorectal cancer syndromein a human individual comprising the steps of (a) isolating DNA from thehuman individual; (b) assaying the DNA using multiplex PCR formicrosatellite loci associated with colorectal cancer or hereditarynon-polyposis colorectal cancer syndrome relative to a normal humanindividual (c) diagnosing colorectal cancer or hereditary non-polyposiscolorectal cancer syndrome in the human individual based on thefrequency of microsatellite loci. In an aspect of the invention themicrosatellite loci that are assayed include BAT26, D17S250, MYC-L, oneor both of BAT40, and optionally one or more of BAT25, D5S346, D2S123,ACTC, D10S197 and D18S55. In aparticular embodiment ofthe invention twoPCR reactions are used to assay the microsatellite loci, and theamplicons or extension products are analyzed using automated fragmentanalysis. The diagnostic method facilitates a determination of theoptimum treatment regimen for the individual.

[0022] In an embodiment of a method of the invention at least 2,preferably at least 4, 6, 8, or 10 microsatellite loci are detected. Ina particular embodiment 11 microsatellite loci are detected.

[0023] The method of the invention enables the identification ofdifferent types of tumors (e.g. colorectal tumor and other tumors)including MSI-H tumors (high or >40% frequency of MSI among the panel ofmicrosatellite loci, i.e. MSH+), MSI-L tumors (low or <40% of MSI), andMSS (microsatellite stable) tumors (Boland, R et al, Cancer Res.58:5248-570). LOH tumors may also be identified using a method of theinvention. LOH or “Loss of Heterozygosity” refers to an allelicimbalance where an allele is lost/reduced in the tumor when comparedwith its expression in matched normal cells.

[0024] In colorectal cancer, individuals with MSI-H tumors have a betteroutcome than those with MSI-L or MSS tumors. MSI-High indicates a changein the mismatch repair pathway with probable inactivation of themismatch repair genes, hMSH2 or hMLH1. Further screening of hMSH2 orhMSLH1 may be carried out, and where there is a strong family historygermline mutation screening of mismatch repair genes may be undertaken.In individuals where there is no family history, hypermethylation of thehMLH1 promoter region may be analyzed.

[0025] These and other aspects, features, and advantages of the presentinvention should be apparent to those skilled in the art from thefollowing drawings and detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The invention will now be described in relation to the drawingsin which:

[0027]FIG. 1 is a schematic diagram of a method of the invention.

[0028]FIG. 2 shows sensitivity detection of MSI at mononucleotiderepeats and MSI at dinucleotide repeats.

[0029]FIG. 3 is a comparison of a MSI analysis of mononucleotide repeatsof a colorectal tumor and a normal subject using an automated multiplexmethod of the invention and a manual radioactive method.

[0030]FIG. 4 is a comparison of a MSI analysis of dinucleotide repeatsof a colorectal tumor and a normal subject using an automated multiplexmethod of the invention and a manual radioactive method.

[0031]FIG. 5 shows the results of an analysis of MSI-High tumor with thepanel 1 loci (Table 1).

[0032]FIG. 6 shows the results of an analysis of MSI-High tumor with thepanel 2 loci (Table 2).

[0033]FIG. 7 shows the results of an analysis of a LOH tumor with panel1 loci (Table 2).

DETAILED DESCRIPTION OF THE INVENTION

[0034] In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See for example, Sambrook, Fritsch, & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); DNA Cloning:A Practical Approach, Volumes I and II (D. N. Glover ed. 1985);Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic AcidHybridization B. D. Hames & S. J. Higgins eds. (1985); Transcription andTranslation B. D. Hames & S. J. Higgins eds (1984); Animal Cell CultureR. I. Freshney, ed. (1986); Immobilized Cells and enzymes IRL Press,(1986); and B. Perbal, A Practical Guide to Molecular Cloning (1984).

[0035] Glossary

[0036] The term “individual” refers to any mammal, particularly humans.

[0037] The term “amplicon” or “amplified product” refers to a discreetamplification product synthesized in an amplification reaction (e.g. PCRreaction) and corresponding regions that is intended to be amplified ina method of the invention.

[0038] The term “microsatellite instability” or “microsatelliteanalysis” refers to the measurement or detection of alterations inmicrosatellite sequences which are known to those skilled in the art torepresent a specific pattern of genomic instability caused by DNAmismatch repair defects. Alterations in microsatellite sequences areclinically useful in the diagnosis and monitoring of various types ofcancer. Microsatellites are short tandem repeat sequences that arebroadly distributed in a genome.

[0039] The term “amplify” or “amplification” refers to a process bywhich nucleotide sequences are amplified in number. There are severalwell known means for enzymatically amplifying nucleotide sequences (seereview in BioTechnology 8:290-3, 1990). The most commonly used method isthe Polymerase Chain Reaction (PCR). PCR employs a thermostable DNApolymerase, known primer sequences, and heating cycles that separate thereplicating DNA strands and exponentially amplify a nucleotide sequenceof interest. The PCR process is fully described in Erlich et al, Science1991 252:1643-50, M. Innis, et al Science 1988, Science 239:

[0040]487-91). Other amplification systems include ligase chain reaction(LCR), and nucleic acid sequence-based amplification (NASBA). Theinvention is not limited to any particular amplification system, sinceother systems may be developed which would benefit by the practice ofthe invention.

[0041] A “mulitplex polymerase chain reaction” is a polymerase chainreaction wherein more than one region of target DNA is amplifiedsimultaneously in a single reaction vessel.

[0042] The term “detectable reporter label” or “label” refers to amolecule that is incorporated indirectly or directly into anoligonucleotide primer of the amplified product. A label moleculefacilitates the detection of an oligonucleotide which becomes part of anamplified DNA sequence. Suitable labels include chromophores,fluorescent molecules, enzymes, antigens, heavy metals, magnetic probes,dyes, radioactive materials, phosphorescent groups, chemiluminescentmoieties, and electrochemical detecting moieties.

[0043] A label may be incorporated directly into an oligonucleotide byusing a deoxynucleoside triphosphate (dNTP) containing a label in theprocess of synthesizing the oligonucleotide. Alternatively, the labelmay be incorporated indirectly into an oligonucleotide by coupling aprimer at the 5′ end with a linker (e.g. aminohexyl linker) using astandard DNA synthesis cycle and coupling a label such as a fluorescentdye-NHS ester via the linker. Fluorescent molecules that are useful forlabeling an oligonucleotide are known to those skilled in the art. Suchmolecules may include amine-reactive groups that are reactive to endterminal amines of an oligionucleotide, sulfonyl chlorides that areconjugated to an oligonucleotide through amine residues, and likemolecules. A fluorescent molecule may be attached by covalent ornon-covalent means. Protocols for incorporating fluorescent moleculesare described for example in Kamik, et al, 1995 Hum. Mol. Genet.4:1889-1894.

[0044] The term “primer” refers to an oligonucleotide capable of actingas a point of initiation for DNA synthesis when annealed to acomplimentary sequence under suitable conditions, and in the presence ofnucleotide triphosphosphates. The primers can be in the form ofribonucleotides, deoxyribonucleotides, modified ribonucleotides,modified deoxyribonucleotides, modified phosphate-sugar backboneoligonucleotides, nucleotide analogs, and mixtures thereof.

[0045] The term “sample” refers to any body tissue or fluid suitable fordetecting tumor cells, including biopsies, bone marrow aspirates, lymphnode aspirates, effusions, ascites, cerebrospinal fluid, and peripheralblood. The sample is preferably a nucleic acid sample from the abovetissues or fluids suitable for detecting tumor cells. Methods forpreparing nucleic acid samples are well known to those skilled in theart. The concentration of nucleic acids in the samples to be used in thepresent invention may be about 20-75 ng, more preferably 25 to 50 ng,most preferably 20-25 ng.

[0046] In the amplification steps of the present invention a PCRreaction may be employed which utilizes primer sets for at least four(preferably at least 6, 8, or 10) selected microsatellite lociassociated with cancer, particularly mismatch-repair deficient tumors,preferably sporadic colorectal and hereditary non-polyposis colorectalcancer syndrome. The primers are selected so that they are suitable forhydridization on complementary strands of a corresponding targetmicrosatellite loci to permit formation of a polymerase chain reactionextension products. There is a mismatch which interferes with formationof such an extension product when the primers hybridize to any othernucleotide sequence present in the sample. The concentration of theforward primers in the PCR reaction mixture may be about 25 to 65 ng,and the concentration of the reverse primers in the PCR reaction mixturemay be about 30 to 840 ng. The PCR extension products in a particularset may be distinguished from other PCR extension products in differentsets. The primers are blended with the sample to form a polymerase chainreaction mixture. The mixtures are subjected to one or more polymerasechain reaction cycles involving a denaturation treatment, ahydridization treatment, and an extension treatment. In thehybridization treatment the target specific portion of a primer ishybridized to the target microsatellite loci. In the extensiontreatment, the hybridized primers are extended to form extensionproducts complementary to the target nucleotide sequence to which theprimer is hybridized. Detailed process conditions for carrying out theamplification steps are set out in the protocols in the Example.

[0047] After the reaction mixture is subjected to the PCR cycles, thelabeled extension products are detected. This indicates that presence ofone or more target microsatellite sequences in the sample. Automatedfragment analysis may be used to detect the labeled fragments.

[0048] In accordance with an aspect of the invention, the firstamplification (e.g. PCR) step employs primers for the BAT26 and D17S250loci, and optionally one or more of BAT25, D5S346, D2S123, and ACTC. Inaccordance with another aspect of the invention, the secondamplification step employs primers for MYC-L, one or both of BAT40 andBAT34C4, and optionally one or both of D10S197 and D18S55. Specificmicrosatellite primer pairs used to amplify and detect microsatelliteinstability in accordance with the invention are disclosed in Tables 1and 2 attached hereto. General Information on the loci is set out inTable 5.

[0049] The following non-limiting examples are illustrative of thepresent invention:

EXAMPLE 1

[0050] An efficient diagnostic test was developed for screening of tumorMSI based on a fluorescent multiplex PCR system and automated fragmentanalysis. The assay consists of a panel of 11 microsatellite lociincluding those loci (BAT25, BAR26, D2S123, D5S346 and D17S250)recommended by the National Cancer Institute (Boland R et al, 1997Cancer Res 58:5248-57). The microsatellite panel includes onetetranucleotide, six dinucleotide, and four mononucleotide loci. DNAextracted from paraffin embedded tissue (25ng) is amplified in twomultiplex PCR reactions. (See detailed protocols set out below andTables 3 (1^(st) amplification step) and Table 4 (2^(nd) amplificationstep) setting out various PCR reactions that were carried out todetermine the optimal conditions for a method of the invention.) Thefluorescent labelled PCR amplicons (size range 65-230 bp) are analyzedusing AB1377 GeneScan and Genotyper software. This diagnostic assay wasvalidated by analyzing ten colorectal cancer cases by both fluorescentmultiplex PCR and conventional radioactive labelled PCR and gelelectrophoresis. The assay sensitivity was determined by MSI analysis oftumor DNA serially diluted with matched normal DNA, and was found torange from 10% for mononucleotide loci to 40% for dinucleotide loci(FIG. 2). Overall, this diagnostic assay offers a fast, sensitive, andcost-effective method of MSI detection and is most suitable for highthroughput screening for mismatch-repair deficient tumors.

[0051] Microsatellite Analysis Protocols

[0052]1 ^(ST) Amplification Step

[0053] Normal and Tumor DNA samples arrive from the Biospecimenrepository already purified using the Qiagen Tissue Kit.

[0054] 1.) Dilute DNA to 25 ng from the original concentration.

[0055] 2.) STEP 1 PCR: Set up master mix cocktail with the followingconditions:

[0056] 3.0 ul 10X PCR Buffer

[0057] 0.9 ul 50X MgCl (1.5 mM final)

[0058] 0.6 ul Forward Primer D17S250

[0059] 0.6 ul Reverse Primer D17S250

[0060] 1.2 ul dNTP's (0.4 mM final)

[0061] 0 ul H₂O

[0062] 0.6 ul Taq (3 units final)

[0063] 2.) To each labelled tube add the appropriate DNA (normal ortumor) in the following format:

[0064] 1.0 ul DNA (25 ng final)

[0065] 2.0 ul H₂O

[0066] 3.) With tubes on ice, add 6.9 ul of master mix cocktail to eachtube and pipette up and down a few times to mix.

[0067] 4.) PCR tubes at the following conditions: 94° C. 5 min 94° C. 30sec - - - 40 cycles 55° C. 30 sec - - - 40 cycles 72° C. 30 sec - - - 40cycles 72° C. 10 min  4° C. forever

[0068] 5.) STEP 2 PCR: While the PCR reaction is starting mix theremaining primers in a tube in the following amounts:

[0069] 0.3 ul Forward Primers of ACTC, D5S346 (33 ng each primer)

[0070] 0.6 ul Forward Primers of D2S123, BAT 26, BAT 25 (66 ng eachprimer)

[0071] 0.3 ul Reverse Primer of ACTC (33 ng each primer)

[0072] 0.6 ul Reverse Primer of BAT 26 (66 ng each primer)

[0073] 4.2 ul Reverse Primers of D2S123 and D5S346 (420 ng each primer)

[0074] 8.4 ul Reverse Primer BAT 25 (840 ng each primer)

[0075] 6.) Add 20.1 ul of above mixture to each of the tubes when thereaction has gone through 5 cycles. Continue with PCR program.

[0076] 7.) Dilute PCR product using 7 ul PCR product mixed with 16 ulH₂O

[0077] 8.) Make a master mix of loading dye, formamide and TAMRA in thefollowing amounts:

[0078] 1X

[0079] 4.0 ul formamide

[0080] 1.0 ul Genescan TAMRA

[0081] 0.5 ul loading dye

[0082] 9.) Mix 4.0 ul of diluted PCR product with 5.5 ul of loading dyemix.

[0083] 10.) Load 2.0 ul of above unto a 5% polyacrylamide gel and runfor 2 hours on the ABI 377 Sequencer (3000 volts, 60 milliAmps and 200Watts).

[0084] 11.) Analyze data using GeneScan and Genotyper.

[0085] 2^(ND) Amplification Step

[0086] Normal and Tumor DNA samples arrive from the Biospecimenrepository already purified using the Qiagen Tissue Kit.

[0087] 1.) Dilute DNA to 25 ng from the original concentration.

[0088] 2.) Set up master mix cocktail with the following conditions:

[0089] 2.0 ul 10X PCR Buffer

[0090] 0.6 ul 50X MgCl (1.5 mM final)

[0091] 1.5 ul Forward Primer (0.3 μl each of BAT 40, MYC-L, BAT 34C4,D10S197, D18S55) (33 ng each primer)

[0092] 1.5 ul Reverse Primer (0.3 ul each of BAT 40, MYC-L, BAT34C4,D10S197, D18S55) (33 ng each primer)

[0093] 0.8 ul dNTP's (0.4 mM final)

[0094] 8.2 ul H₂O

[0095] 0.4 ul Taq (2 units final)

[0096] 2.) To each labelled tube add the appropriate DNA (normal ortumor) in the following format:

[0097] 1.0 ul DNA (25 ng final)

[0098] 4.0 ul H₂O

[0099] 3.) With tubes on ice, add 25 ul of master mix cocktail to eachtube and pipette up and down a few times to mix.

[0100] 4.) PCR tubes at the following conditions: 94° C. 5 min 94° C. 30sec - - - 40 cycles 57° C. 30 sec - - - 40 cycles 72° C. 30 sec - - - 40cycles 72° C. 10 min  4° C. forever

[0101] 5.) Dilute PCR product using 7 ul PCR product mixed with 16 μlH₂O

[0102] 6.) Make a master mix of loading dye, formamide and TAMRA in thefollowing amounts:

[0103] 1X

[0104] 4.0 ul formamide

[0105] 1.0 ul Genescan TAMRA

[0106] 0.5 ul loading dye

[0107] 7.) Mix 4.0 ul of diluted PCR product with 5.5 ul of loading dyemix.

[0108] 8.) Load 2.0 ul of above unto a 5% polyacrylamide gel and run for2 hours on the ABI 377 Sequencer (3000 volts, 60 milliAmps and 200Watts).

[0109] 9.) Analyze data using GeneScan and Genotyper

Example 2

[0110] Below is a summary of colorectal cancer cases analyzed using amultiplex MSI panel and their MSI status. PANEL MSI-H MSI-L MSS TOTAL  I(6 loci) 76 2 170 248 II (11 loci) 2 12 0 14

[0111] TABLE 1 First Panel of Markers for Evaluation of MSI inColorectal Cancer PRODUCT MONO OR 5′ FLUORESCENT LOCUS PRIMER SEQUENCESIZE DINUCLEOTIDE TAG BAT 25 Forward-TCG CCT CCA AGA ATG TAA GT 110-125Mononucleotide HEX Reverse-TCT GCA TTT TAA CTA TGG CTC BAT 26Forward-TGA CTA CTT TTG ACT TCA GCC 107-125 Mononucleotide TETReverse-AAC CAT TCA ACA TTT TAA ACC C D17S250 Forward-GAA GTG ATG AAAAGT AAT TGA TC 190-230 Dinucleotide FAM Reverse-GCT GGC CAT ATA TAT ATTTAA ACC D5S346 Forward-ACT CAC TCT AGT GAT AAA TCG GG 110-135Dinucleotide FAM Reverse-AGC AGA TAA GAC AGT ATT ACT AGT T D2S123Forward-AAA CAG GAT GCC TGC CTT TA 200-230 Dinucleotide TET Reverse-GGACTT TCC ACC TAT GGG AC ACTC Foward-CTT GAC CTG AAT GCA CTG TG 70-98Dinucleotide FAM Reverse-ATT CCA TAC CTG GGA ACG AG

[0112] TABLE 2 Second Panel of Markers for Evaluation of MSI inColorectal Cancer PRODUCT MONO OR 5′ FLUORESCENT LOCUS PRIMER SEQUENCESIZE DINUCLEOTIDE TAG BAT 40 Forward-ATT AAC TTC CTA CAC CAC AAC 110-140Mononucleotide HEX Reverse-GTA GAG CAA GAG CAC CTT G MYG-L Forward-TGGCGA GAG TCC ATC AAA G 140-210 Tetranucleotide HEX Reverse-CCT TTT AAGCTG CAA CAA TTT G BAT 34G4 Forward-ACC CTG GAG GAT TTC ATC TC 120-145Mononucleotide TET Reverse-AAC AAA GCG AGA CCC AGT CT D10S197Forward-ACC ACT GCA CTT CAG GTG ACA 155-185 Dinucleotide TET Reverse-GTGATA CTG TCC TCA GGT CTC C D18S55 Forward-GGG AAG TCA AAT GCA AAA TG135-165 Dinucleofide FAM Reverse-AGC TTG TGA GTA ATC TTA TGC TGT G

[0113] TABLE 3 ATTEMPT # BAT 25 BAT 26 D17S250 D2S123 D5S346 ACTCMULTIPLEX 25ng and 50 ng DNA Same PCR conditions as FIRST ATTEMPT 1.5 mMfinal MgCl above-tried annealing at 55 BAT 25, BAT 26, 33 ng PrimerD17S250, D2S123, 0.4 mM final DNTP' Tried multiplex conditions D5S346 2units Taq 25 ng DNA 1.5 mM final MgCl ACTC Alone 96 10 min 33 ng Primer96 10 sec - - - 45 cycles 0.4 mM final DNTP's 55 30 sec - - - 45 cycles2 units Taq 70 30 sec - - - 45 cycles Amplified very well 70 10 min 4forever All amplified well except Primers all amplified very well underthese condition on their own. D17S250, D2S123 and D5S346 were a littlelow MULTIPLEX 25 ng DNA 25 ng DNA 25 ng DNA 25 ng DNA 25 ng DNA 25 ngDNA SECOND ATTEMPT 1.5 mM final MgCl 1.5 mM final MgCl 1.5 mM final MgCl1.5 mM final MgCl 1.5 mM final MgCl 1.5 mM final MgCl 66 ng Primer F/840ng R 33 ng Primer 45 ng Primer 33 ng Primer 33 ng Primer 33 ng PrimerINCREASED F/R 0.4 mM final DNTP's 0.4 mM final DNTP's 0.4 mM finalDNTP's 0.4 mM final DNTP's 0.4 mM final DNTP's 0.4 mM final DNTP'sPRIMERS FOR 2 units Taq 2 units Taq 2 units Taq 2 units Taq 2 units Taq2 units Taq BAT 25 Tried extra forward and extra Same PCR conditions asSame PCR conditions as forward and reverse Same PCR conditions as SamePCR conditions as Same PCR conditions as ADDED ACTC TO above above aboveabove above MULTIPLEX Same PCR conditions as above Amplified very wellAmplified very well Didn't amplify Amplified weaker than the Amplifiedweaker than the Amplified very well rest rest MULTIPLEX 25 ng DNA 25 ngDNA 25 ng DNA 25 ng DNA 25 ng DNA 25 ng DNA THIRD ATTEMPT 1.5 mM finalMgCl 1.5 mM final MgCl 1.5 mM final MgCl 1.5 mM final MgCl 1.5 mM finalMgCl 1.5 mM final MgCl 66 ng Primer F/840 ng R 33 ng Primer 33 ng Primer33ng Primer 33 ng Primer 33 ng Primer BAT 25 WITH ALL 0.4 mM finalDNTP's 0.4 mM final DNTP's 0.4 mM final DNTP's 0.4 mM final DNTP's 0.4mM final DNTP's 0.4 mM final DNTP's OTHER PRIMERS 2 units Taq 2 unitsTaq 2 units Taq 2 units Taq 2 units Taq 2 units Taq EXCEPT D17S250 SamePCR conditions as Same PCR conditions as Same POR conditions as Same PCRconditions as Same PCR conditions as Same PCR conditions as above aboveabove above above above BAT 25 WITH Amplified very well in Amplifiedvery well in Amplified well in multiplex Amplified well in multipleAmplified well in multiplex mult multiplex Did not amplify with BAT 2Slightly weaker than rest Slightly weaker than rest MULTIPLEX 25 ng DNA25 ng DNA 25 ng DNA 25 ng DNA 25 ng DNA FOURTH ATTEMPT 1.5 mM final MgCl1.5 mM final MgCl 1.5 mM final MgCl 1.5 mM final MgCl 1.5 mM final MgCl66 ng Primer F/840 ng R 33 ng Primer 33 ng Primer F/420 ng R 33 ngPrimer F/420 ng R 33 ng Primer NO D17S250 0.4 mM final DNTP's 0.4 mMfinal DNTP's 0.4 mM final DNTP's 0.4 mM final DNTP's 0.4 mM final DNTP's2 units Taq 2 units Taq 2 units Taq 2 units Taq 2 units Taq INCREASEDPRIMER Same PCR conditions as Same PCR conditions as Same PCR conditionsas Same PCR conditions as Same PCR conditions as RATIO TO 14:1 FORD2S123 AND D5S346 above above above above above D2S123 and D5S346amplifed much better than previously seen. All other loci amplified verywell. FIFTH MULTIPLEX 25 ng DNA 25 ng DNA 25 ng DNA 25 ng DNA 25 ng DNAATTEMPT 1.5 mM final MgCl 1.5 mM final MgCl 1.5 mM final MgCl 1.5 mMfinal MgCl 1.5 mM final MgCl 66 ng Primer F/840 ng R 33 ng Primer F/33ng R 33 ng Primer F/420 ng R 33 ng Primer F/ 420ng R 33 ng Primer F/33ng R CHANGED TO 0.4 mM final DNTP's 0.4 mM final DNTP's 0.4 mM finalDNTP's 0.4 mM final DNTP's 0.4 mM final DNTP's PLATINUM TAQ 3 units Taq3 units Taq 3 units Taq 3 units Taq 3 units Taq USING MJ PCR 94 5 min 945 min 94 5 min 94 5 min 94 5 min MACHINE 94 30 sec - - - 40 cycles 94 30sec - - - 40 cycles 94 30 sec - - - 40 cycles 94 30 sec - - - 40 cycles94 30 sec - - - 40 cycles 55 30 sec - - - 40 cycles 55 30 sec - - - 40cycles 55 30 sec - - - 40 cycles 55 30 sec - - - 40 cycles 55 30sec - - - 40 cycles 72 45 sec - - - 40 cycles 72 45 sec - - - 40 cycles72 45 sec - - - 40 cycles 72 45 sec - - - 40 cycles 72 45 sec - - - 40cycles 72 10 min 72 10 min 72 10 min 72 10 min 72 10 min 4 forever 4forever 4 forever 4 forever 4 forever All loci are amplifiying very wellFINAL MULTIPLEX 25 ng DNA 25 ng DNA 25 ng DNA 25 ng DNA 25 ng DNA 25 ngDNA CONDITIONS 1.5 mM final MgCl 1.5 mM final MgCl 1.5 mM final MgCl 1.5mM final MgCl 1.5 mM final MgCl 1.5 mM final MgCl 66 ng Primer F/840 ngR 66 ng Primer F/66 ng R 66 ng Primer F/66ng R 66 ng Primer F/420 ng R33 ng Primer F/420 ng R 33 ng Primer F/33 ng R INCREASED BAT26 0.4 mMfinal DNTP's 0.4 mM final DNTP's 0.4 mM final DNTP's 0.4 mM final DNTP's0.4 mM final DNTP's 0.4 mM final DNTP's F/R PRIMER CONC. 3 units Taq 3units Taq 3 units Taq 3 units Taq 3 units Taq 3 units Taq ADDED D175250TO 94 5 min 94 5 min 94 5 min 94 5 min 94 5 min 94 5 min MULTIPLEX 94 30sec - - - 35 cycles 94 30 sec - - - 35 cycles 94 30 sec - - - 40 cycles94 30 sec - - - 35 cycles 94 30 sec - - - 35 cycles 94 30 sec - - - 35cycles 55 30 sec - - - 35 cycles 55 30 sec - - - 35 cycles 55 30sec - - - 40 cycles 55 30 sec - - - 35 cycles 55 30 sec - - - 35 cycles55 30 sec - - - 35 cycles INCREASED D2S123 72 30 sec - - - 35 cycles 7230 sec - - - 35 cycles 72 30 sec - - - 40 cycles 72 30 sec - - - 35cycles 72 30 sec - - - 35 cycles 72 30 sec - - - 35 cycles F PRIMERCONC. 72 10 min 72 10 min 72 10 min 72 10 min 72 10 min 72 10 min 4forever 4 forever 4 forever 4 forever 4 forever 4 forever TWO STEP PCRAll loci are amplifying very well

[0114] TABLE 4 ATTEMPT # BAT 40 BAT 34C4 D10S197 D18S55 MYC-L MULTIPLEX25 ng DNA FIRST 1.5 mM final MgCl ATTEMPT 33 ng F/R Primer BAT 40, 0.4mM final DNTP's BAT34C4 D10S197, 2 units Taq D18S55 MYC-L 94 5 minIncreased 94 30 sec - - - 40 cycles elongationc 55 30 sec - - - 40cycles to 45 sec 72 45 sec - - - 40 cycles 72 10 min 4 forever Allprimers amplified well together, slight background for BAT 40 MULTIPLEX25 ng DNA SECOND 1.5 mM final MgCl ATTEMPT 33 ng Primer Increased 0.4 mMF/R Primer annealing temperature to 57 2 units Taq 94 5 min 94 30sec - - - 40 cycles 57 30 sec - - - 40 cycles 72 45 sec - - - 40 cycles72 10 min 4 forever All primers amplified well, background for BAT 40 isgreatly diminished FINAL 25 ng DNA 25 ng DNA 25 ng DNA 25 ng DNA 25 ngDNA MULTIPLEX 1.5 mM final MgCl 1.5 mM final MgCl 1.5 mM final MgCl 1.5mM final MgCl 1.5 mM final MgCl CONDITIONS 33 ng Primer 33 ng Primer 33ng Primer 33 ng Primer 33 ng Primer USING 0.4 mM F/R Primer 0.4 mM F/RPrimer 0.4 mM F/R Primer 0.4 mM F/R Primer 0.4 mM F/R Primer PLATINUMTAQ 2 units Taq 2 units Taq 2 units Taq 2 units Taq 2 units Taq USING MJPCR 94 5 min 94 5 min 94 5 min 94 5 min 94 5 min MACHINE 94 30 sec - - -40 cycles 94 30 sec - - - 40 cycles 94 30 sec - - - 40 cycles 94 30sec - - - 40 cycles 94 30 sec - - - 40 cycles 57 30 sec - - - 40 cycles57 30 sec - - - 40 cycles 57 30 sec - - - 40 cycles 57 30 sec - - - 40cycles 57 30 sec - - - 40 cycles 72 45 sec - - - 40 cycles 72 45sec - - - 40 cycles 72 45 sec - - - 40 cycles 72 45 sec - - - 40 cycles72 45 sec - - - 40 cycles 72 10 min 72 10 min 72 10 min 72 10 min 72 10min 4 forever 4 forever 4 forever 4 forever 4 forever All loci areamplifyIng very well

[0115] TABLE 5 LOCI LOCATION GENE NEAR MARKER TYPE OF REPEAT SIZE FIRSTMULTIPLEX PANEL BAT 25 4q12 Intron of the C-kit TTTT.T.TTTT.(T)₇.A(T)₂₅110-125 oncogene BAT 26 2p Intron 5 of MSH2 (T)₅........(A)₂₆ 105-125D5S346 5q21-22 APC-a tumour (CA)₂₈ 110-140 suppressor gene D2S123 2p1 6MSH2 (CA)₁₃TA(CA)₁₅(T/G A)₇ 200-230 ACTC 15q11-qter ACTC (GT)₂₄  65-100D17S250 17q11.2-q12 BRCA1 (TA)₇...............(CA)₂₄ 190-230 SECONDMULTIPLEX PANEL BAT 40 1p13.1 Intron of 3-B-hydroxysteroidTTTT.TT.(T)₇..........TTTT.(T)₄₀ 100-135 dehydrogenase gene BAT34C4 11thexon of p53-repeat is (T)₃C(T)₆C(T)₁₇C(T)₅C(T)₃ 120-145 in 3′non-translated region D10S197 10qter CACCAGA(CA)₇.A.A.(CA)₁₂(AGAAA)₂155-185 D18S55 18q22.1 DCC-Deleted in Colorectal (CA)₂₃ 135-165Carcinomas MYCL 1p32 MYCL GAAAA(GAAA)₂TAAA(A/G)₁₀ 140-210GAAAGA(GAAA)₁₄GAAA(GAAAA)₈ GAAAAA(GAAAA)₃

[0116] The present invention is not to be limited in scope by thespecific embodiments described herein, since such embodiments areintended as but single illustrations of one aspect of the invention andany functionally equivalent embodiments are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

[0117] All publications, patents and patent applications referred toherein are incorporated by reference in their entirety to the sameextent as if each individual publication, patent or patent applicationwas specifically and individually indicated to be incorporated byreference in its entirety. All publications, patents and patentapplications mentioned herein are incorporated herein by reference forthe purpose of describing and disclosing the cell lines, vectors,methodologies etc. which are reported therein which might be used inconnection with the invention. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

[0118] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

We claim:
 1. A method for evaluating microsatellite instability in atumor sample by detecting microsatellite loci in the sample comprising:(a) forming a polymerase chain reaction mixture comprising the tumorsample, a polymerase and primer sets for at least two selectedmicrosatellite loci associated with cancer, each primer setcharacterized by (a) a forward primer containing a sequencecomplimentary to a 5′ upstream primer-specific portion of a selectedmicrosatellite loci; and (b) a reverse primer complementary to a 3′downstream primer-specific portion of the same microsatelite loci,wherein one of the primers has a detectable reporter label; (b)subjecting the polymerase chain reaction mixture to polymerase chainreaction cycles to form amplified products complementary tomicrosatellite loci sequences in the tumor sample; (c) detecting thereporter labels and distinguishing the amplified products to indicatethe presence of one or more of the microsatellite loci in the sample;and (d) repeating steps (a) to (c) with primer sets for at least twodifferent selected microsatellite loci.
 2. A method as claimed in claim1 wherein in step (b) the polymerase chain reaction cycles comprise adenaturation treatment, wherein hybridized nucleic acid sequences areseparated, a hybridization treatment, wherein the primers hybridize totheir complementary primer-specific portions of a microsatellite locisequence, and an extension treatment, wherein the hybridized primers areextended.
 3. A method as claimed in claim 1 wherein in step (a) theprimers are for the BAT26 and D17S250 loci, and optionally one or moreof BAT25, D5S346, D2S123, and ACTC loci.
 4. A method as claimed in claim1 wherein in step (a) the primers are the primers in Table
 1. 5. Amethod as claimed in claim 1 wherein in step (b) the primers are forMYC-L, one or both of BAT40 and BAT34C4, and optionally one or both ofD10S197 and D18S55.
 6. A method as claimed in claim 1 wherein in step(b) wherein the primers are the primers in Table
 2. 7. A method asclaimed in claim 1 wherein the detectable reporter label is achromophore, fluorescent molecule, enzyme, antigen, heavy metal,magnetic probe, dye, radioactive material, phosphorescent group,chemiluminescent moiety, or electrochemical detecting moiety.
 8. Amethod as claimed in claim 1 wherein the tumor sample is a body tissueor fluid suitable for detecting tumor cells.
 9. A method as claimed inclaim 1 wherein the tumor sample comprises nucleic acids.
 10. A methodas claimed in claim 9 wherein the nucleic acids are present in the tumorsample at a concentration of 20-75 ng.
 11. A method as claimed in claim1 wherein the concentration of the forward primer is about 25-65 ng andthe concentration of the reverse primer is about 30-840 ng.
 12. A methodas claimed in claim 1 wherein the cancer involves defects in mismatchrepair of genes.
 13. A method as claimed in claim 1 wherein the canceris leukemia, colorectal cancer, breast cancer, lung cancer, prostatecancer, brain tumors, central nervous system tumors, bladder tumors,melanomas, liver cancer, bone cancer, testicular carcinoma, ovariancarcinoma, head and neck tumors, or cervical cancer.
 14. A method asclaimed in claim 1 wherein the cancer is colorectal cancer or hereditarynon-polyposis colorectal cancer syndrome.
 15. A method for diagnosingcolorectal cancer or hereditary non-polyposis colorectal cancer syndromein a human individual comprising the steps of (a) isolating DNA from thehuman individual; (b) assaying the DNA using multiplex polymerase chainreaction for microsatellite loci associated with colorectal cancer orhereditary non-polyposis colorectal cancer syndrome relative to a normalhuman individual (c) diagnosing colorectal cancer or hereditarynon-polyposis colorectal cancer syndrome in the human individual basedon the frequency of microsatellite loci.
 16. A method as claimed inclaim 15 wherein at least 6, 8, or 10 microsatellite loci are assayed.17. A method as claimed in claim 15 wherein the microsatellite lociassayed are BAT26, D17S250, MYC-L, one or both of BAT40, and optionallyone or more of BAT25, D5S346, D2S123, ACTC, D10S197 and D18S55.
 18. Amethod as claimed in claim 15 wherein the microsatellite loci assayedare the loci identified in Table 1 and Table
 2. 19. A kit comprisingcompositions selected from the group consisting of primers and ancillaryreagents used in a polymerase chain reaction in a method as claimed inclaim
 1. 20. A kit comprising compositions selected from the groupconsisting of primers and ancillary reagents used in a mulitplexolymerase chain reaction in a method as claimed in claim 15.